In Vivo Neuronal Migration Assay with MAP6 Depletion Protocol
Phase 1: MAP6 shRNA Viral Production and Pilot Knockdown (Days 1-21)
shRNA Design: Design 3 independent shRNAs targeting MAP6 (MISSION shRNA, Sigma-Aldrich): target sequences in 3' UTR and coding sequence. Include scrambled control shRNA with no homology to mouse genome. Clone into AAV2/9 vector under H1 promoter.
Viral Production: Co-transfect HEK293T cells (10 cm dishes, 80% confluent) with shRNA AAV plasmid, pAAV2/9 helper, and pXR5 rep/cap using PEI (1:3 ratio). Harvest at 72 hours, concentrate via iodixanol gradient (40/60% cushion). Titer via qPCR (genome copies/mL, target ≥10¹² GC/mL).
...In Vivo Neuronal Migration Assay with MAP6 Depletion Protocol
Phase 1: MAP6 shRNA Viral Production and Pilot Knockdown (Days 1-21)
shRNA Design: Design 3 independent shRNAs targeting MAP6 (MISSION shRNA, Sigma-Aldrich): target sequences in 3' UTR and coding sequence. Include scrambled control shRNA with no homology to mouse genome. Clone into AAV2/9 vector under H1 promoter.
Viral Production: Co-transfect HEK293T cells (10 cm dishes, 80% confluent) with shRNA AAV plasmid, pAAV2/9 helper, and pXR5 rep/cap using PEI (1:3 ratio). Harvest at 72 hours, concentrate via iodixanol gradient (40/60% cushion). Titer via qPCR (genome copies/mL, target ≥10¹² GC/mL).
Pilot Knockdown Verification: Transduce primary cortical neurons (DIV 5) with AAV-shMAP6 or AAV-scramble at MOI 5. Assess knockdown at DIV 10, 14, 18 via qRT-PCR (MAP6 #Mm01238749_m1, normalized to GAPDH) and western blot (anti-MAP6, 1:500, Abcam #ab199937). Select shRNA with ≥70% knockdown and ≤20% off-target effects.
Phase 2: In Utero Electroporation and Migration Analysis (Days 22-49)
In Utero Electroporation: Anesthetize E14.5 pregnant C57BL/6J mice (Crawford Long Hospital). Expose uterine horns, inject AAV-shMAP6 (or AAV-scramble, 1 μL, + 0.025% Fast Green dye) into lateral ventricle of each embryo. Apply electric pulses (5 pulses, 50 ms, 950 mV, 1 Hz) via CUY21EDIT electroporator with forceps electrodes.
Tissue Collection: At E18.5 and P0, P7, harvest brains from electroporated pups (identified by GFP co-expression from IRES-eGFP cassette). Fix in 4% PFA (24 hours, 4°C), cryoprotect in 30% sucrose, embed in OCT.
Migration Analysis: Cut coronal sections (20 μm) on cryostat. Immunostain for GFP (1:500, Abcam #ab13970) to identify electroporated neurons. Counterstain with DAPI. Image on Vectra 3.0 (10× objective). Quantify: (a) distribution of electroporated neurons across cortical layers (layer I through VIb), (b) distance traveled from ventricular zone, (c) morphology (leading process length, branch points).
Phase 3: Functional Consequences and Rescue (Days 50-70)
Cortical Lamination Assessment: In P7 brain sections, assign electroporated neurons to cortical layers based on DAPI staining patterns and Ctip2/Satb2 immunohistochemistry (layer 5/6b markers). Compare distribution between shMAP6 and scramble groups.
MAP6 Rescue Experiment: Generate AAV-shMAP6-resistant MAP6 cDNA (silent mutations in shRNA target site). Co-electroporate shMAP6 + rescue construct. Assess whether MAP6 overexpression rescues migration phenotype (confirms specificity).
Cell Survival Analysis: At P7, quantify apoptotic neurons in electroporated population via cleaved caspase-3 immunostaining (1:200, Cell Signaling #9661). Calculate survival percentage relative to scramble controls.